1
IUPUI Multidisciplinary Undergraduate Research Institute (MURI) Mentorship Project Proposal Form COVER PAGE Date of submission: March 2, 2009 Proposed project Title: Ability of Tobacco to Increase the Virulence of Oral Disease by Affecting Human Cells and Bacteria Principal mentor’s name: Richard L. Gregory, Ph.D. Principal mentor’s title, phone number, e-mail, department, and school: Professor, 274-9949,
[email protected], Department of Oral Biology, School of Dentistry Co-mentor’s name, title, phone number, e-mail, department, and school (repeat for multiple comentors): L. Jack Windsor, Ph.D., Associate Professor, 274-1448,
[email protected], Department of Oral Biology, School of Dentistry Fengyu Song, D.D.S., Ph.D., Assistant Professor, 274-2415,
[email protected], Department of Oral Biology, School of Dentistry The project will be carried out and completed (check only one): Academic year Summer (Note: The academic year ends May 1)
(Summer runs June 1 – Aug 1)
This is a block grant project but not carried out in a class. (Note: Block grant projects involve more than five students. The requirement for multidisciplinarity is not as stringent for this type of proposal but, nevertheless, preference will be given to proposals that include multidisciplinary teams.)
This is a block grant project for a class, designated as an IUPUI MURI project. (Note: Block grant proposals to be carried out within a class require the signature of the department chair. Block grant projects involve more than five students. The requirement for multidisciplinarity is not as stringent for this type of proposal but, nevertheless, preference will be given to proposals that include multidisciplinary teams.)
Total number of students requested: 5 (Note: The total number of students must exceed the number of mentors)
Total Number of freshmen and/or sophomores to be recruited: 2 (Note: Preference will be given to projects that include freshmen and sophomores)
Disciplines or majors of students (at least two disciplines or majors): Biology, Chemistry, PreDental, Pre-Medical, SPEA, Liberal Arts Skills expected from students: Good analytical skills
2
PROPOSAL Ability of Tobacco to Increase the Virulence of Oral Disease by Affecting Human Cells and Bacteria Objectives: Our overall objectives of this proposal are identical to our 2008 MURI projects, however, individual projects (see below) will differ significantly. The Indiana Tobacco Prevention and Cessation Agency 2006 Annual Report reported that the smoking rate for adults in Indiana recently increased from 24.9% in 2004 to 27.3% in 2005. This resulted in Indiana being presently ranked number two behind Kentucky in the United States with the highest smoking rates. The addictive component of cigarettes, nicotine, is the most widely studied component of tobacco in terms of its effect on various cell populations of the oral cavity. Oral tissues of smokers are exposed to high doses of nicotine through either saliva or crevicular fluid. In the gingival crevicular fluid of smokers, nicotine concentrations are nearly 300 times those found in plasma (20 ng ⁄ml). Nicotine binds to the root surface in smokers. This can alter gingival and periodontal ligament fibroblast attachment and proliferation in vitro. Reports have also demonstrated reduced collagen production, as well as increased collagenase activity in gingival fibroblast cultures exposed to nicotine. Furthermore, cigarette smoke negatively impacts bone healing in animal models. This could reflect the ability of nicotine to stimulate osteoclastic differentiation, impair angiogenesis, and/or inhibit expression of various growth factors. Taken together, these factors seemingly contribute to the increased tissue destruction and diminished healing response in smokers, which becomes particularly problematic for procedures involving tissue regeneration. Tobacco use impacts coronary heart disease, lung cancer, oral cancers, pancreas cancer, kidney cancer, urinary bladder cancer, chronic obstructive pulmonary disease, chronic bronchitis, emphysema, pneumonia, dental caries, periodontal disease, osteoporosis, sleep problems, cataracts, and thyroid disease (Grave’s Disease). In addition, tobacco exposure is a recognized risk factor for a number of oral diseases. In the oral cavity, cigarette smoking is a known risk factor for periodontal disease, alveolar bone loss and tooth loss while smokeless tobacco usage is a risk factor for oral cancer and gingival recession. With a wide variety of substances present in tobacco and tobacco smoke, any one of them could potentially participate in these disease processes. Nicotine is a major suspect in the above disease processes. Furthermore, the prevalence of dental caries is higher among smokers than non-smokers. However, the mechanism is not clear especially whether tobacco components have effects on oral bacteria. Preliminary data indicate that nicotine upregulates several Streptococcus mutans genes associated with attachment to tooth surfaces facilitating caries or to endothelial cells in heart tissue causing atherosclerosis or endocarditis. Secondly, several authors have reported growth inhibitory and toxic effects of nicotine at different concentrations on human periodontal ligament fibroblasts as determined by various growth or viability assays. In addition, nicotine has adverse effects on mouse molar tooth buds during tooth development in organ culture. The molecular mechanism of this phenomenon is far from clear. Recently, the impact of nicotine on the wound healing process involving matrix metalloproteinases (MMPs) has been demonstrated and is controversial. Studies will focus on the effects of nicotine or tobacco smoking condensate on human pulp fibroblasts, oral mucosal epithelium cells and S. mutans. All student projects will be focused on the effects of tobacco or nicotine on human pulp fibroblasts, oral mucosal epithelium cells and oral bacteria and will involve a multidisciplinary approach involving biochemistry, cell biology and molecular biology (Song and Windsor), and microbiology/ immunology (Gregory). Any human studies will obtain the appropriate IRB approval before any
3
work is conducted on human subjects, data or tissues. Each study may require independent IRB applications. Projects using human cell culture lines will receive or already have received either IRB exemption or expedited review. These labs are very familiar with IRB applications and in fact specific studies that students may elect to work on may have IRB approval. Research Methodology: Student research projects will focus on the effects of nicotine on: 1) adhesins, metabolism and biofilm formation of S. mutans (Gregory lab); 2) expression of proinflammatory cytokines from human gingival fibroblasts (Windsor lab); and 3) release of MMP from human pulp fibroblasts and ability of green tea to inhibit (Song lab). 1. Bacterial studies in the Gregory lab include nicotine effects on growth using biofilm formation and bacterial metabolism. Examples include examining the effect of nicotine on specific live/dead bacteria staining and microscopy, up- or down-regulation of S. mutans genes in biofilm formation and effect on binding to extracellular matrix proteins associated with endothelial cells, adherence of biofilm organisms to solid surfaces using microplate assays, and metabolic pathways in biofilm bacteria focusing on phosphotransferase activity. 2. The MURI students in the Windsor laboratory this semester has focused on the effects of smoke condensate on the expression of monocyte chemoattractant protein-1 (MCP-1) and growth-regulated oncogene-alpha (GROα) from human gingival fibroblasts. The students started out learning how to grow gingival fibroblasts including how to sub-culture and count them. They are now focusing on experiments examining the expression of MCP-1 and GROα from the cells after exposure to smoke condensate utilizing Western blot analyses and ELISA. Increased levels of both GROα and MCP-1 have been show to be related to periodontal disease and smoking has been shown to be a risk factor for periodontal disease. 3. The preliminary data from the Song lab has suggested that both nicotine and cigarette smoke condensate (CSC) affect the biological activities of human pulp fibroblast (HPF). Nicotine and CSC alternate HPF’s extracellular matrix remodeling ability through matrix matalloproteinase (MMP) pathway, and also influence HPF’s cytokine expression. These changes may result in the future tissue damage or improperly repairmen seen in smoking population. How to prevent, inhibit and even reverse these processes caused by smoking will be the critical to treat the smoking related diseases and bring insight into the future treatment and prevention strategy. Green tea and their active components have long been suggested to be anti-oxidants and function to prevent and even reverse tissue damages caused by super oxidants. If the green tea will slow down or even reverse the effects of smoking is still under investigation. In the proposed research project, green tea extract will be added with nicotine or CSC to HPFs to test the HPF ability to express MMP and various cytokines. Zymography and cytokine array will be employed as major techniques for this project. Based on the knowledge gained from the above projects, we will advance our research to investigate the effects of tobacco components and products on human cells and oral bacteria. These effects include proliferation, toxicity, cancer induction (especially oral cancer), extracellular matrix binding and degradation, expression and activation of the MMPs, and enhanced oxidative stress. These multiple effects of tobacco and its components on human cells results in multiple possible research projects. These projects include how tobacco and its components affect the oral cavity, p53 mutation, and oxidative stress, as well as how these effects might be counteracted by herbal products and their active ingredients. Team Organization:
4
a. The Principal Mentor will take the lead in supervising students to initially identify the lab and specific project they will work on and to coordinate the team meetings and ensure student attendance at all required research seminars and programs; b. The Principal and Co-Mentor(s) will be available to provide students assistance in design of specific studies and experiments, to meet regularly with each student to plan experiments and analyze data and prepare research reports and presentations; c. Graduate students and postdocs will train students in some of the laboratory assays; d. One or two of students in Biology, Pre-Dental, Pre-Medical, SPEA or Liberal Arts will take the leadership in role in the Gregory lab to accomplish the biofilm experiments; e. One or two each of students in Chemistry, Pre-Dental, Pre-Medical, SPEA or Liberal Arts will take the leadership in role in the Windsor or Song labs to accomplish the cytokine or matrix metalloproteinase assays, respectively. These studies will complement others ongoing in the Tobacco Cessation and Biobehavioral Signature Center. Students will work closely with the three MURI faculty (Drs. R.L. Gregory, L.J. Windsor and F. Song) as well as with postdoctoral and Ph.D. students working on other tobacco-related projects. Students will meet individually with their MURI faculty at least 2-3 times a week to discuss experimental design and research data. In addition, all students and MURI faculty will meet once a month for each student to present his/her project and data. This will build camaraderie among the MURI students and provide them with a certain level of comfort in discussing their project to a group. Students approved for this project will be given background readings specific to the laboratories they will primarily be working in (see references 1-7 as examples) before their MURI project actually begins. Students from various backgrounds (Science-Biology, Chemistry, Pre-Dental and Pre-Medical, SPEA, Liberal Arts) are desired as it allows a diversification of ideas. Many SPEA students would benefit because of their potential interest in environmental and health affairs connected with the focus on tobacco usage in this project. Accepting students into this project from non-science backgrounds (Liberal Arts) allows their experience to grow and for them to understand a different discipline. This will allow the students to begin to understand the topic they will be investigating. Expected Outcomes: Attempts have been made to understand the mechanism(s) of tobacco and its smoke how influences human oral cells and oral microorganisms. The project is designed to systemically analyze those effects from different perspectives and be able to combine the results of the oral cells and oral bacteria together to have a complete picture regarding the tobacco’s effects on oral cavity. The results from this proposed project will be helpful to support ongoing projects in Tobacco Cessation and Biobehavioral Signature Center, which are pilot studies needed for NIH grant applications in the near future. In addition, students will acquire sufficient knowledge to be conversant in their specific field of research and master the necessary skills to conduct research and present their work with the platform provided from this proposed project in dental school’s research environment. All students will be expected to present their research at all required MURI and/or UROP workshops, as well as our school’s annual Research Day held in April every year. We will ask the students to participate in Research Day even though they would have completed their research in the summer prior to Research Day because many of the students applying to our laboratories for research projects are pre-dental students. All students conducting research at our school participate in a biweekly Student Research Presentation Program (SRPP) and the MURI Fellows would be expected to present their work at the end of the summer research period. The projects conducted during the summer on these projects will
5
significantly differ from the other MURI projects in that these individual experiments will be different such as using different human cell types or examining different S. mutans enzyme activities that were not examined in the 2008 projects. For example, one possible fall/spring project will be: effect of nicotine on S. mutans binding to extracellular matrix proteins. Benefits: With the knowledge gained from this proposed project, information for understanding how tobacco affects the oral cavity can be obtained. Building on this knowledge, future strategies for intervention with tobacco on oral pathological condition can be constructed. This proposed project will also help to build the base for future NIH grant applications. Moreover, students in this program would benefit by gaining important research skills specific to their projects as well as interact with the MURI faculty and other associated research personnel (graduate students and technicians). One of the tangible benefits will be that each student will be able to record at least one published abstract (MURI workshop and/or IUSD Research Day) on their resume and able to verbally present their work before a group of faculty and other students. Time Table (see table below): In the first week students would be introduced to all personnel in the laboratory, department and to other MURI faculty and students, assigned desk space and a lab coat and take required online training courses offered by IUPUI in laboratory safety and blood borne disease without regard to the type of research to be conducted (see time line table). Also in the first week, students will be given additional reading material on their topic and receive detailed instruction in the basic aspects of the laboratory procedures. Subsequent weeks will be devoted to experimental design and pilot experiments. By the second month of the project, students should be fairly independent in the experimental procedures they have been instructed on and conducted preliminary pilot experiments. It is anticipated that the bulk of the research data that will be reported will be obtained in the latter half of the 9 week program. The last week of the program will be devoted to completion of any remaining experiments and writing the final report of the project. In addition, students will prepare a poster throughout the entire program with the introduction and methods sections being written in the early weeks of the project and the results and discussion sections being prepared at the end of the program.
ACTIVITY Introduction to Lab Proc. Learn Exp. Procedures Pilot Exp. Actual Exp. Completion of Exp. Written Report
1 X
ACTIVITIES DURING EACH WEEK of PROGRAM 2 3 4 5 6 7 8 X X
X
X
X
X
X X
X
X
X
9
X X X
Itemized Budget: Total $2,000. The supply budget consists of an equal division of the funds among each student (for 5 students, each would be able to utilize up to $400/each for supplies or small equipment).
6
Bibliography: 1. Zheng, C., and R. Gregory. 2008. The effect of nicotine and cotinine on the growth of Streptococcus mutans and the protein expression. Abstracts of the American Society for Microbiology. 2. Gregory, R.L., J.C. Kindle, L.C. Hobbs, T. VanTo and H.S. Malmstrom. 1990. Effects of smokeless tobacco on the ability of secretory component to bind to the IgA/J chain complex. Human Antibodies and Hybridomas 1:126-131. 3. Gregory, R.L., J.C. Kindle, L.C. Hobbs and H.S. Malmstrom. 1991. Effect of smokeless tobacco use on mucosal immune factors. Archs. Oral Biol. 36:25-31. 4. Zhou, J., B.L. Olson, and L.J. Windsor. 2007. Nicotine increases the collagen-degrading ability of human gingival fibroblasts. J. Periodont. Res. 42:228-235. 5. Almasri, A., K. Wisithphrom, L.J. Windsor, and B.L. Olson. 2007. Nicotine and lipopolysaccharide affect cytokine expression from gingival fibroblasts. J. Periodont. 78:533-541. 6. Song, F., A.S. Bergdoll, and L.J. Windsor. 2006. Temporomandibular joint synovial fibroblasts mediate serine proteinase dependent Type I collagen degradation. Biochim. et Biophys. Acta. 1760:1521-1528. 7. Song, F., K. Wisithphrom, J. Zhou, and L.J. Windsor. 2006. Matrix metalloproteinase dependent and independent collagen degradation. Frontiers in Bioscience. 11:3100-3120. Short Resumes: See attached 2 page NIH-style biosketches for Drs. Richard Gregory, L. Jack Windsor and Fengyu Song in Appendix. Support Letters: None, no collaborative arrangements. Appendix: Please see attached from MURI projects in Summer 2008 and Academic Year 200809 and mentor and co-mentor biosketches. Please check below as appropriate and provide the study number or, if not available, indicate when it will be provided, if the research involves the use of: Animals (IACUC Study #): _________________ Humans (IRB Study #): _Only if needed, to be obtained with initiation of specific studies; some are already approved (i.e., 0304-58 and exempt human fibroblast and pulp cell studies)_______________ r-DNA (IBC Study #): _____________________ Human Pathogens, Blood, Fluids, or Tissues must be identified if used: ______
SIGNATURES Name and Signature of the Principal Mentor: (writing the full name suffices as signature for electronic copies)
_Richard L. Gregory________________________________________March 2, 2009__ Name Signature Date
7
APPENDIX
All students participating and funded by the MURI Program in Summer 2008 and academic year 2008-09 either have presented their research data or will be in the next 2 months at the following meetings (see abstracts below). To be presented at the 87th annual meeting of International Association of Dental Research General Session and Exhibition/ 38th Annual Meeting of the American Association of Dental Research/ 33rd Annual Meeting of Canadian Association of Dental Research to be held at the Miami Beach Convention Center in Miami, Florida, from April 1-4, 2009 The Effects of Cigarette Smoke Condensate on Human Pulp Cells E. GROW1, J. SUN2, N. SANTOSH2, L.J. WINDSOR3, R.L. GREGORY3, and F. SONG2, 1Department of Chemistry Indiana University Purdue University, Indianapolis, IN, 2Indiana University School of Dentistry, Indianapolis, IN, 3Indiana University, Indianapolis, IN Cigarette smoke condensate (CSC) is produced when tobacco is burned and it is composed of more than 4,000 chemicals. It is well known that smoking tobacco can cause lung cancer and other health problems such as periodontal disease and oral cancer. However, very little is known about the effects of CSC on cell repairing and regeneration ability. Objectives: This research project examined the effects of CSC on human pulp cells (HPC) and is our first step to investigate smoking's effect on HPC repairing and regeneration ability. Materials and Methods: HPCs were collected from dental patients whose teeth were extracted for orthodontic treatment with Institutional Review Board approval. HPCs (50,000 cells/well) were exposed to various concentrations of CSC (0, 3.125, 6.25, 12.5, 50, 100, 200 and 400µg/ml) for three days. The cytotoxicity and cell viability effects of the CSC on HPCs were evaluated by performing lactate dehydrogenase (LDH, Roche Applied Science, Mannheim, Germany) and water soluble tetrazolium-1 (WST-1, Roche Applied Science) assays, respectively. To test their collagen degrading ability under the influence of CSC, HPCs (100,000 cells/well) were seeded as a single colony in each well of Type I collagen-coated 6-well plates and supplemented with CSC (0, 12.5, 25, and 400µg/ml) for 1, 3 and 5 days before staining with Coomassie blue. The conditioned media were also collected to test for proteinase activity utilizing zymography. Results: The CSC was toxic and inhibited HPC proliferation at 400µg/ml. Concentrations of CSC at 12.5 and 25µg/ml stimulated the HPC ability to degrade collagen. The production of pro- and active MMP-2 was increased in the cultured media. Conclusion: CSC is toxic to HPCs and affects pulp cell growth. CSC at non-toxic concentrations changes the HPC ability to remodel extracellular matrix, in part, by producing more MMP-2.
THE EFFECTS OF NICOTINE ON OSTEOBLASTS Elizabeth F. Smith, Jun Sun, Fengyu Song, and L. Jack Windsor, Department of Oral Biology, Indiana University School of Dentistry, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202
8
Tobacco use is a significant risk factor for the formation and progression of periodontal disease, as well as multiple types of cancer. Periodontal disease involves the destruction of bone and eventually tooth loss. Nicotine is a major component of tobacco and is a very addictive substance. The effects that nicotine has on bone-forming cells (osteoblasts) have not been well studied. Objective: Therefore, the purpose of this study was to examine the effects of nicotine on osteoblast proliferation, viability, cell-mediated collagen degradation, and expression of multiple cytokines/growth factors. Methods: Osteoblasts (MG63) (American Type Culture Collection, Manassa, VA) were grown in low glucose Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum at 37o and in 5% CO2. Osteoblast proliferation was measured by the water-soluble tetrazolium-1 (WST-1) assay (Roche Diagnostics GmbH, Mannheim, Germany) and viability was measured by the lactate dehydrogenase (LDH) assay (Roche Diagnostics GmbH, Mannheim, Germany) after the osteoblasts were exposed to different concentrations of nicotine (0-1000 µg/ml). The results were statistically analyzed (ANOVA, Tukey’s, p
